The ultimate goal of this project is to make a high resolution map of the Tetrahymena genome. Tetrahymena thermophila is a unicellular eukaryote of great value as an experimental system due to its rapid growth, its structural and functional differentiations, its accessibility to combined genetic and molecular experimental approaches, its large evolutionary distance from other commonly used genetic model systems, its potential for biotechnological applications, and the safety with which it can be handled. A good genetic map has become an acute need for Tetrahymena work. Our initial objective is to identify and map 500 DNA polymorphisms. Polymorphic STSs (sequence-tagged sites) will at once be detected and molecularly cloned using the RAPD (randomly amplified polymorphic DNA) PCR method. We will construct a linkage map using a hierarchical mapping scheme based on meiotic and mitotic recombination. Assuming random distribution of the 500 DNA polymorphisms, we calculate a >99% probability that any new mutation (or cloned DNA segment) will fall within 30 cM (and thus within readily detectable linkage) of a previously mapped DNA polymorphism. We also propose to map these polymorphisms, by genetic and molecular means, to the autonomously-replicating DNA pieces of the macronucleus. In addition to a generally useful map of DNA polymorphisms, we expect to refine our estimates of the frequency of meiotic recombination, and to measure the frequency micronuclear mitotic and macronuclear somatic recombination; to confirm in detail the physical colinearity of macronuclear autonomously replicating pieces with micronuclear chromosomes; to locate the centromeres of each MIC chromosome, and put ourselves in a favorable position for ultimately investigating a ciliate centromere; to develop a strong foundation for building a physical map of the Tetrahymena genome; to facilitate cloning novel genes by reducing the complexity of the DNA to be searched, or by allowing short chromosome walks from a mapped and physically available DNA segment. With suitable modifications, methodology to be developed here may also be useful for mapping the human genome and other complex genomes of interest.